Targeted therapy, specifically through kinase inhibition, continues to emerge as a method for personalized therapy in non-small cell lung cancer (NSCLC). As an example, patients with driver mutations in the epidermal growth factor receptor (EGFR) can be treated with Erlotinib or other targeted EGFR inhibitors to improve their outcomes. However, many lung cancer patients (~50%) have tumors that do not have known oncogenic driver mutations; subsequently, their treatment options are limited. In order to expand the arsenal of therapeutic agents for these patients, quantitative measurements of protein phosphorylation and kinase activity levels will be used to examine the dominant signaling pathways in each tumor, when compared with the corresponding normal lung tissue resected from the same lobe of the lung. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) will be used to monitor more than 260 tyrosine-phosphorylated peptides to generate phosphorylation profiles and more than 160 peptides labeled and affinity purified using activity-based protein profiling to indicate the levels of active kinases. Together, these data will be used to indicate activated pathways and infer hubs that can be disrupted using existing kinase inhibitors. This quantitative platform for patient assessment will seek options for treatment of NSCLC with no known driver mutations, with the ultimate goal of defining a small list of candidate biomarkers that can be tested in biopsies. The development of this systematic approach to explore signaling in lung cancer will also provide insights into the analysis of signaling networks with quantitative mass spectrometry and the clinical utility of LC-MRM.